rXXXX American Chemical Society 3027 DOI: 10.1021/jz100967z | J. Phys. Chem. Lett. 2010, 1, 3027–3032 pubs.acs.org/JPCL Crowding Effects on Amyloid Aggregation Kinetics Andrea Magno, Amedeo Caflisch,* and Riccardo Pellarin* Department of Biochemistry, University of Z€ urich, Winterthurerstrasse 190, CH-8057 Z€ urich, Switzerland ABSTRACT Biological protein self-assembly occurs in the cellular milieu, densely occupied by other macromolecules which do not participate directly in the aggregation process. Excluded volume effects arising in such a crowded environ- ment deeply affect the thermodynamics and kinetics of biological processes, like protein folding, ligand binding, and protein aggregation. Here, Langevin dynamics simulations of a simplified model of an amphipathic polypeptide are used to investigate how macromolecular crowding influences the amyloid aggregation kinetics. The simulations show that the net influence of macromolecular crowding on the self-assembly process is the result of two competing effects: oligomer stabilization and solution viscosity increase. Notably, the net effect crucially depends on the aggregation propensity and pathways. Therefore, comparative studies of concentration and crowding effects on the kinetics of amyloid aggrega- tion could shed light on the underlying self-assembly mechanism. SECTION Biophysical Chemistry A myloid fibrils are ordered polypeptide aggregates that have been related to several neurodegenerative patho- logies, such as Alzheimer's, Parkinson's, Huntington's and prion diseases, 1,2 and, more recently, also to biological functionalities. 3,4 These findings have paved the way to a wide range of experimental and computational studies aimed at understanding the details of the fibril formation mechanism. Most of these investigations were usually performed in ideal homogeneous conditions, though the actual cellular milieu is a much more complex environment. Several studies have pointed out the effects of geometric confinement on fibril formation and protein folding. 5,6 A universal property of the cells is that they are crowded. 7,8 Indeed, it has been estimated that 20-30% of the cell cytoplasm is occupied by proteins, RNA, membranes, polysaccharides, and several organelles. 9 Although the concentration of every species is low, these macromolecules exclude a significant fraction of the total available volume. 10 The nonspecific excluded volume effect is expected to sensibly affect all biological reactions in which proteins are involved. 11 Using scaled particle theory, 12 Minton and Ellis have predicted that macro- molecular crowding dramatically increases the association rate of proteins and the relative stability of the unfolded and native state. 13,14 Computational studies have shown that the presence of inert, repulsive cosolutes stabilizes the native, compact state of proteins 15,16 and that the presence of nano- particles is able to catalyze amyloid aggregation. 17 Experi- ments 18-21 conducted with large and weakly interacting macromolecules such as polyethyleneglycol (PEG) and Fycoll have confirmed these theoretical predictions, showing that the excluded volume effect is able to modify the subtle equilibrium between the folded, functional state and aberrant structures prone to aggregation. Earlier, a very simple coarse-grained model of an aggrega- tion-prone, amphipathic peptide was developed to investigate the kinetics of ordered aggregation. 22 The peptide monomer has a single degree of freedom, and the relative free-energy profile has only two minima, corresponding to the aggrega- tion-prone and aggregation-protected states. By varying a single parameter of the model, that is, by reducing the β-aggregation propensity, the roughness of the free-energy landscape and the heterogeneity of the fibril elongation pathway increase. In previous simulation studies, heteroge- neous kinetics of aggregation and multiple pathways were observed in bulk solution and in the presence of lipid bilayers. At high amyloidogenic conditions, the process of fibril formation is downhill and fast, whereas at low amyloi- dogenic conditions, several intermediates are detected, and the nucleation occurs through a micellar oligomer. 23 It has also been pointed out that amyloidogenicity determines the effect on peptide aggregation of the presence of lipid bilayer; while aggregation-prone peptides fibrillate faster by adsorbing on the bilayer surface, the ordered self-assembly of poorly amyloidogenic peptides is hindered by the vesicles. 24,25 In this work, a spherical model of softly repulsive crowders is used, together with the simple model of the amphipathic peptide, to investigate amyloid aggregation kinetics at different concen- trations of crowders. It is found that the influence of the crowders has a pronounced dependence on the amyloidogenic tendency of the peptide. The simulations were performed with 125 peptides and a number of softly repulsive crowders ranging from 250 to 5000 (see Methods and Table 1). Snapshots of the simulation Received Date: July 16, 2010 Accepted Date: September 12, 2010